Neurons regulate when and where they synthesize new proteins to maintain basal and activity-dependent compartmental fidelity. Control of this process is largely regulated at translational initiation which typically occurs at the 5'-most AUG codon in an appropriate mRNA sequence context. Emerging data in simple model systems suggests that un-annotated, upstream open reading frames (uORFs) are utilized on a significant fraction of all transcripts. These uORFs often initiate at near-AUG (one base different from AUG) codons and display condition-dependent usage, suggesting they may play a regulatory role in translational control. How such atypical initiation events are mediated and what roles they play in neuronal function and disease are unknown. The central hypothesis of this grant is that upstream open reading frame usage is an important regulator of translational dynamics in neurons, and that proteins produced by translation from un-annotated uORFs will contribute to neuronal function. Our group recently discovered a novel mechanism for protein translational initiation associated with nucleotide repeats in the neurodegenerative disorder Fragile X-associated Tremor Ataxia Syndrome (FXTAS). FXTAS results from a CGG nucleotide repeat expansion in the 5' untranslated region (UTR) of the FMR1 gene. We discovered that this repeat expansion elicits initiation at a near-AUG codon in the 5'UTR, resulting in translation through the repeat via a process known as RAN (Repeat Associated Non-AUG) translation. This translation event leads to synthesis of a poly-glycine containing protein, FMRpolyG, even at normal repeat sizes. This proposal will first determine when FMRpolyG is synthesized in neurons both basally and in response to activity. Further, the impact of FMRpolyG synthesis on translation of the major FMR1 gene product, FMRP- a protein implicated in autism and intellectual disability will be assessed. To determine the prevalence of similar uORF utilization events in neurons, the novel next-generation sequencing technique known as ribosomal profiling will be used to measure ribosomal occupancy within 5'UTRs across the neuronal transcriptome. These studies will employ cellular imaging, biochemical and bioinformatics techniques to explore a new area in neurobiology with relevance to both synaptic plasticity as well as numerous neurological diseases.